DNA/RNA Concentration & Purity Calculator
Quantify nucleic acids using absorbance readings at 260 nm and assess purity from 260/280 and 260/230 ratios. Includes conversion factors for dsDNA, ssDNA, RNA, and oligonucleotides.
Parameters
For 260/280 ratio (purity check)
For 260/230 ratio (contamination check)
Results
| Parameter | Value |
|---|---|
| Sample Type | dsDNA |
| Conversion Factor | 50 µg/mL per A₂₆₀ |
| A₂₆₀ | 0.500 |
| A₂₈₀ | 0.270 |
| A₂₃₀ | 0.100 |
| Dilution Factor | 1× |
| Path Length | 1 cm |
| Concentration | 25.00 ng/µL |
Understanding Nucleic Acid Quantification
Nucleic acids (DNA and RNA) absorb ultraviolet light strongly at 260 nm due to their purine and pyrimidine ring systems. By measuring absorbance at this wavelength and applying the Beer-Lambert Law, we can calculate concentration. Proteins absorb strongly at 280 nm, so comparing ratios tells us about purity.
The Calculation
Conversion factors are empirical and depend on nucleotide composition: dsDNA (50 µg/mL per A₂₆₀), ssDNA (33 µg/mL), RNA (40 µg/mL), and oligos (33 µg/mL). Since 1 µg/mL = 1 ng/µL, the concentration in ng/µL is numerically identical to µg/mL.
The 260/280 Ratio: Protein Contamination
Pure DNA has a 260/280 ratio of ~1.8; pure RNA is ~2.0. Proteins have high absorption at 280 nm due to aromatic amino acids (Trp, Tyr, Phe), so a low 260/280 ratio indicates protein contamination. Common sources: phenol (from extraction), residual lysozyme, or incomplete precipitation. Values outside 1.7–2.0 (DNA) or 1.8–2.2 (RNA) should trigger concern.
The 260/230 Ratio: Salt & Carbohydrate Contamination
Ideal 260/230 ratios are 2.0–2.2. Low ratios indicate contamination by guanidinium salts (from lysis buffers used in extraction), carbohydrates (from cells or extraction reagents), or residual phenol. These compounds absorb at 230 nm. Re-precipitation with ethanol or additional wash steps can improve this ratio. Values below 1.8 warrant investigation and re-purification.
Microfluidics: On-Chip Nucleic Acid Quantification
In microfluidic channels with short optical path lengths (50–500 µm), the reduced absorbance requires either higher concentrations, more sensitive detectors, or longer on-chip optical paths (e.g., serpentine or integrated Z-cells) to achieve reliable quantification. Some chips use fluorescence-based methods (e.g., intercalating dyes like SYBR Green or high-sensitivity fluorescence) for improved sensitivity over absorbance alone. On-chip quantification enables rapid, low-volume sample analysis in point-of-care and high-throughput applications.
Limitations & Tips
- • Conversion factors assume typical genomic or synthetic DNA/RNA; composition-biased sequences may differ slightly
- • Extremely high absorbances (>2.0) are outside the linear range of most spectrophotometers; dilute your sample
- • Always blank your cuvette or instrument with the same buffer used to dissolve your sample
- • Keep samples at 4 °C to minimize degradation and evaporation
- • Store samples in low-binding tubes (e.g., LoBinding or LoBind) to prevent nucleic acid loss
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